Fig. 4
Engineered Synechocystis sp. PCC 6803 strains with four copies of kivdS286T significantly improved isobutanol (IB) and 3-methyl-1-butanol (3M1B) titers compared to control strain with three copies of kivdS286T. A Schematic diagram of plasmids used to generate strains in Fig. 4. P5 is a self-replicating plasmid; P6-P13 are integrative plasmids targeting various sites of Synechocystis chromosome. B The relative IB titer of engineered Synechocystis strains HX56, HX61-63, HX78 and HX80. C Simplified pathway for IB and 3M1B biosynthesis. Endogenous enzymatic reactions are written in black; heterologous enzymatic reactions are written in red; knock-out/knock-down enzymatic reactions are written in grey. Abbreviations of enzymes: KivdS286T, α-ketoisovalerate decarboxylase (Lactococcus lactis); PEPc (encoded by sll0920), phosphoenolpyruvate carboxylase; PDH (encoded by slr1934 and sll1721), pyruvate dehydrogenase E1 component; Ddh (encoded by slr1556), D-lactate dehydrogenase; LeuA (encoded by slr0186 and sll1564), 2-isopropylmalate synthase. Abbreviations of intermediates: PEP, phosphoenolpyruvate. D The relative 3M1B titer of engineered Synechocystis strains HX56, HX61-63, HX78 and HX80. E Western-immunoblot analysis of all expressed enzymes. L, ladder (in kDa). Ten micrograms, 20 μg, and 20 μg of total soluble proteins were loaded for each strain to detect Strep-tagged, Flag-tagged, and His-tagged proteins, respectively. Protein size: KivdS286T, 61 kDa. F Growth profile of engineered Synechocystis strains HX56, HX61-63, HX78 and HX80. Results are the mean of three biological replicates, each with three technical replicates. Error bars represent standard deviation. Asterisk represents significant difference between engineered strains and control strain (one-way ANOVA, *p < 0.05, **p < 0.005)